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. 2020 Nov 24;10(12):1596. doi: 10.3390/biom10121596

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© 2020 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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LA25 cells stained for total Cx43 and pY247 (A) and pS279/282 (B, arrows point to yellow, apparently internalized vesicles or connexosomes). In (C–E), LA25 cells expressing either Cx43-mEmerald (green) or HaloTag (red) were mixed resulting in gap junctions and connexosomes containing both colors (plasma membrane marker is shown in grayscale). In D, pY247 Ab plus Cx43-HaloTag yields purple indicating phosphorylation of Cx43-HaloTag at gap junctions (arrows) and in connexosomes in cytoplasm (arrowheads) while in E, pS279/282 occurs extensively in connexosomes (arrowheads indicate overlay of green, red, and blue so it appears white). Bars are 10 µm. Quantification, shown in (F), shows proportion of Cx43 signal on connexosomes versus gap junctions in BWEM and LA25 cells.